The interaction of cells with their extracellular matrix in the in vivo environment plays a crucial role in the organization, homeostasis, and function of tissues and organs. Continuous communication between cells and their surrounding extracellular matrix environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, the development of form (morphogenesis), angiogenesis, wound healing, and even tumor metastasis. Both biochemical and biophysical signals from the extracellular matrix modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death.
In turn, the cell can modify its extracellular matrix environment by modulating the synthesis and degradation of specific matrix components. The realization of the significance of cell-extracellular matrix interaction has led to a renewed interest in characterizing extracellular matrix constituents and the basic mechanisms of cell-extracellular matrix interaction.
Various basement membrane tissues and other extracellular matrix tissues can be utilized as tissue graft constructs for remodeling tissues in vivo or for in vitro applications. Complex scaffolds representing combinations of extracellular matrix components in a natural or processed form are commercially available and can also be used for remodeling tissues in vivo or for in vitro applications. For example, extracellular matrices such as Human Extracellular Matrix (Becton Dickinson) and MATRIGEL® are commercially available. Basement membrane tissues and other extracellular tissues, such as submucosa tissues, harvested from warm blooded vertebrates have also shown great promise as unique graft materials for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation of cell populations in vitro.
For example, submucosa tissue constructs are characterized by excellent mechanical properties, including high compliance, high tensile strength, a high burst pressure point, and tear-resistance, while offering additional advantages such as resistance to infection, lack of immunogenicity, and stability. Furthermore, submucosa tissues can be extracted or fluidized or components can be purified from submucosa tissues to provide compositions useful in tissue graft applications. Extracts or fluidized preparations or purified extracellular matrix components can be utilized as additives for tissue culture media to promote in vitro cell growth and proliferation, and can also be used as active ingredients for other tissue graft compositions, such as wound healing compositions.
As a tissue graft material, submucosa tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. Numerous studies have shown that submucosa tissue is capable of inducing host tissue proliferation, remodeling, and regeneration of tissues following implantation in a number of in vivo environments, including the urinary tract, the body wall, tendons, ligaments, bone, cardiovascular tissues, and other vascular tissues, and the central nervous system. Submucosa tissue has been used successfully, for example, in vascular grafts, for urinary bladder repair, for hernia repair, for replacement and repair of tendons and ligaments, for body wall repair, as a vaginal sling, for rotator cuff repair, for wound care and management, and as a dermal graft. Upon implantation of the submucosa tissues, cellular infiltration and rapid neovascularization are observed and the submucosa materials are remodeled into host replacement tissue with site-specific structural and functional properties.
Accordingly, submucosa tissue can be used as a tissue graft construct, for example, in its native form, in its fluidized form, in the form of an extract, or as components extracted from or solubilized from submucosa tissue and subsequently purified. The fluidized forms of vertebrate submucosa tissue can be gelled to form a semi-solid composition that can be implanted as a tissue graft construct or utilized as a cell culture substrate. As a tissue graft material, the fluidized form of submucosa tissue can be injected, or delivered using other methods, to living tissues to enhance tissue remodeling. Furthermore, the fluidized form can be modified, or can be combined with specific proteins, growth factors, drugs, vectors, or other therapeutic agents for promoting the enhancement or suppression of tissue remodeling at the site of injection. Moreover, the fluidized form of submucosa tissue can be combined with cells, for example primary cells or cell lines, prior to injection to further enhance the repair or replacement of diseased or damaged tissues.
Applicants have discovered here that the physical state of an extracellular matrix graft construct and molecular composition should be considered in the design of new and improved graft constructs. Modifying the conditions used to form a collagen-based matrix allows for the controlled alteration of the micro-structural and subsequent mechanical properties of the resulting engineered matrix. Furthermore, the micro-structural and mechanical properties of the engineered matrix impact cell behavior including proliferation, migration, and differentiation of cells growing on or within the matrix. The engineered matrices of the present invention are distinguished from previously prepared amorphous aggregates of collagen because the engineered matrices described herein comprise collagen fibrils with specific characteristics, including, but not limited to, a specific fibril area fraction (i.e., density) and/or a specific elastic or linear modulus (i.e., stiffness). The engineered matrices described herein can also be made under conditions, where, for example, collagen concentration is controlled.
In one illustrative aspect, a kit is provided. The kit comprises a three-dimensional, engineered matrix comprising collagen fibrils wherein the fibril area fraction of the matrix is about 7% to about 26%. In various illustrative embodiments, the matrix comprises purified collagen or solubilized extracellular matrix components, the matrix is lyophilized, the kit comprises instructional materials for use of the matrix wherein the instructional materials include instructions for injection of the matrix into a patient and/or instructions for growth of cells on the matrix, and the matrix further comprises particulate extracellular matrix material for bulking. In other illustrative kit embodiments, the solubilized extracellular matrix components comprise components from vertebrate submucosa tissue, or basement membrane tissue.
In another embodiment a kit is provided. The kit comprises a three-dimensional, engineered matrix comprising collagen fibrils wherein the elastic or linear modulus of the matrix is about 0.5 to about 40 kPa. In various illustrative embodiments, the matrix comprises purified collagen or solubilized extracellular matrix components, the matrix is lyophilized, the kit comprises instructional materials for use of the matrix wherein the instructional materials include instructions for injection of the matrix into a patient and/or instructions for growth of cells on the matrix, and the matrix further comprises particulate extracellular matrix material for bulking. In other illustrative kit embodiments, the solubilized extracellular matrix components comprise components from vertebrate submucosa tissue, or basement membrane tissue.
In yet another embodiment, a kit is provided for preparing three-dimensional engineered matrices comprising collagen fibrils. The kit comprises a vessel comprising purified collagen or solubilized extracellular matrix components, a glucose solution, a calcium chloride solution, an acid solution selected from the group consisting of HCl, formic acid, sulfuric acid, ethanoic acid, carbonic acid, nitric acid, or phosphoric acid, and a neutralizing solution.
In still another illustrative embodiment, a method of enhancing the repair of tissues in a warm-blooded vertebrate is provided. The method comprises the steps of providing a three-dimensional, engineered matrix comprising collagen fibrils wherein the fibril area fraction of the matrix is about 7% to about 26%, and injecting the matrix into a desired site of said vertebrate.
In another aspect, a method of enhancing the repair of tissues in a warm-blooded vertebrate is provided. The method comprises the steps of providing a three-dimensional, engineered matrix comprising collagen fibrils wherein elastic or linear modulus of the matrix is about 0.5 to about 40 kPa, and injecting the matrix into a desired site of said vertebrate.
In another embodiment, a method is provided of preparing a three-dimensional, engineered matrix comprising collagen fibrils wherein the fibril area fraction of the matrix is about 7% to about 26%. The method comprises the steps of providing purified collagen or solubilized extracellular matrix components, and polymerizing the purified collagen or solubilized extracellular matrix components into fibrils by systematically varying the polymerization conditions. In this embodiment, the polymerization conditions are selected from the group consisting of pH, phosphate concentration, temperature, buffer composition, ionic strength, the solubilized extracellular matrix components polymerized, and concentration of the solubilized extracellular matrix components.
In another embodiment, a method is provided of preparing a three-dimensional, engineered matrix comprising collagen fibrils wherein the elastic or linear modulus of the matrix is about 0.5 to about 40 kPa. The method comprises the steps of providing purified collagen or solubilized extracellular matrix components, and polymerizing the purified collagen or solubilized extracellular matrix components into fibrils by systematically varying the polymerization conditions. In this embodiment, the polymerization conditions are selected from the group consisting of pH, phosphate concentration, temperature, buffer composition, ionic strength, the solubilized extracellular matrix components polymerized, and concentration of the solubilized extracellular matrix components.